Insecticidal compounds and methods for selection thereof

ABSTRACT

A potent and highly specific insecticidal toxin comprising 45 residues and three intramolecular disulfide bonds, as well as a method for selecting potent inhibitors of insect calcium channels.

This application is a Divisional of Ser. No. 09/780,874, filed Feb. 9,2001, now U.S. Pat. No. 6,583,264 which claims priority to U.S.provisional application 60/181,532, filed Feb. 10, 2000, the entirecontents of which are incorporated by reference herein.

1. FIELD OF THE INVENTION

The present invention relates to biological compounds, and genesencoding biological compounds, for use as pesticides, as well as methodsfor obtaining such compounds.

2. BACKGROUND OF THE RELATED ART

Unfortunately, it has increasingly been seen over the past severaldecades that employment of conventional chemical insecticides oftenleads to undesirable environmental consequences. Such consequencesinclude toxicity to non-target organisms such as birds and fish, andhuman health hazards. Furthermore, pesticide management in the UnitedStates and elsewhere in the world is becoming increasingly complicateddue to the evolution of insect resistance to classical chemicalpesticides. Despite over 10 billion dollars being spent each year tocontrol phytophagus insects, global losses in the food supply due toinsects is still estimated to be about 20 to 30 percent (See, Oerke,Estimated crop losses due to pathogens, animal pests and weeds, 72–78 inCrop, production and crop protection: Estimated losses in major food andcash crops (Elsevier, Amsterdam 1994)). There remains, therefore, anurgent need to develop or obtain substances that can be used safely inthe fight against insect pests.

Over the past several years, there have been proposed a number of“environmentally friendly” strategies to combat highly resistant insectpests such as certain species of cotton bollworm (e.g., Helicoverpazea).

One recently introduced approach to insect management is the productionof transgenic crops that express insecticidal toxins, such as engineeredpotato, and cotton crops that express protein toxins from the soilbacterium Bacillus thuringiensis (Estruch, J. J. et al., Transgenicplants: An emerging approach to pest control, Nature Biotechnology 15,137–141, 1997).

A variation of this strategy is the release of insect-specific virusesthat have been genetically engineered to express insecticidalneurotoxins (Cory, J. S. et al., Field trial of a genetically improvedbaculovirus insecticide, Nature 370, 138–140, 1994). Baculoviruses, forexample, are arthropod-specific viruses with no member of thebaculovirus family known to infect either vertebrates or plants. Theinfectivity of some baculoviruses is restricted to a few closely relatedspecies within a single family of lepidopterous insects (moths andbutterflies) (See, e.g., U.S. Pat. No. 5,639,454). Some baculoviruses,such as the beet armyworm nuclear polyhedrosis virus, target only asingle species. As a result of their high degree of specificity,baculoviruses have long been envisaged as potential pest control agentsand were first used as such in the 1970s. Their specificity means thatbaculoviral insecticides complement natural predators, rather thanreplacing them, as is the case with many chemical insecticides. However,to date, baculoviruses have met with only limited commercial success.Most naturally occurring baculoviruses take 4–7 days to kill theirhosts, with some species taking considerably longer. During this timethe insect continues to feed and cause crop damage, thus limiting theability of baculoviral insecticides to compete with chemical agents.

This shortcoming has been addressed by engineering recombinantbaculoviruses that express insect-specific neurotoxins. Expression ofheterologous insect toxins not only reduces the time interval betweenvirus application and insect death, but also reduces the mean feedingtime (Prikhod'ko et al., Effects of simultaneous expression of twosodium channel toxin genes on the properties of baculoviruses asbiopesticides, Biological Control 12, 66–78, 1998). Importantly,introduction of genes for insect-selective toxins does not alter theintrinsic infectivity of the baculovirus or its natural host range(Black et al., Commercialization of baculoviral insecticides, in TheBaculoviruses (ed. Miller, L. K.) 341–387 (Plenum Press, New York, USA,1997)).

New approaches to insect-pest management have stimulated interest inpeptide toxins from the venoms of animals, particularly spiders andscorpions, that prey on insect species.

Zlotkin et al., An Excitatory and a Depressant Insect Toxin fromScorpion Venom both Affect Sodium Conductance and Possess a CommonBinding Site, Arch. Biochem. and Biophysics 240, 877–887, 1985),disclose two insect selective toxins from the venom of the scorpionLeiurus quinqestriatus, one of which induced fast excitatory contractiveparalysis of fly larvae while the other induced slow depressant flaccidparalysis, with both affecting sodium conductance in the neurons.Likewise, Canadian patent 2,005,658 (issued: Jun. 19, 1990 to Zlotkin etal.) discloses an insecticidally effective protein referred to as“LqhP35” derived from the scorpion Leiurus quinquestriatus hebraeus.

A number of investigators have also recognized spider venoms as apossible source of insect-specific toxins for agricultural applications(See, Jackson et al., Ann. Rev. Neurosci. 12, 405–414 (1989)). Forexample, U.S. Pat. No. 4,855,405 (issued: Aug. 8, 1989 to Yoshioka etal.) and U.S. Pat. No. 4,918,107 (issued: Apr. 17, 1990 to Nakajima etal.) both disclose glutamate-receptor inhibitors obtained from the venomof spiders as possible insecticidal agents. In U.S. Pat. No. 5,457,178(issued: Oct. 10, 1995), U.S. Pat. No. 5,695,959 (issued: Dec. 9, 1997),and U.S. Pat. No. 5,756,459 (issued: May 26, 1998), Jackson et al.disclose a family of insecticidally effective proteins isolated from thevenom of the spiders Filistata hibernalis (a common house spider) andPhidippus audax (a “jumping spider”).

A particular group of spiders which has generated considerableinvestigative interest are the funnel-web spiders. WO 89/07608(published: Aug. 24, 1989, Cherksey et al.) discloses low molecularweight factors isolated from American funnel-web spider venoms whichreversibly bind to calcium channels. Adams et al., Isolation andBiological Activity of Synaptic Toxins from the Venom of the Funnel WebSpider, Agelenopsis aperta, in Insect Neurochemistry andNeurophysiology, Borkovec and Gelman (eds.) (Humana Press, New Jersey,1986) teaches that multiple peptide toxins which antagonize synaptictransmission in insects have been isolated from the spider Agelenopsisaperta. In WO 93/15108, a class of peptide toxins known as theω-atracotoxins are disclosed as being isolated from the Australianfunnel-web spiders (Araneae:Hexathelidae:Atracinae) by screening thevenom for anti-Helicoverpa (“anti-cotton bollworm”) activity. Suchtoxins are disclosed to have a molecular weight of approximately 4000amu, to be of 36–37 amino acids in length, and capable of forming threeintrachain disulfide bridges. One of these compounds, designatedω-ACTX-Hv1 has been shown to selectively inhibit insect, as opposed tomammalian, voltage-gated calcium channel currents (Fletcher et al., Thestructure of a novel insecticidal neurotoxin, ω-atracotoxin-Hv1, fromthe venom of an Australian funnel web spider, Nature Struct. Biol. 4,559–566 (1997)). Homologues of ω-ACTX-Hv1 have been isolated from theBlue Mountain funnel-web spider Hadronyche versuta (See, Wang et al.,Structure-function of ω-atrocotoxin, a potent antagonist of insectvoltage-gated calcium channels, Eur. J. Biochem. 264, 488–494 (1999)).

While some insecticidal peptide toxins isolated so far from scorpionsand spiders offer promise, there still remains a significant need forcompounds which display a wide differential in toxicity between insects,and non-insects, and yet which have significant insecticidal activityand a quick action.

3. SUMMARY OF THE INVENTION

The present inventors have isolated, and structurally and functionallycharacterized, a novel insecticidal toxin, designatedω-atracotoxin-Hv2a, from the venom of the Australian funnel-web spiderH. versuta. This toxin is a highly potent and specific antagonist ofinsect calcium channels. The toxin of the present invention shows nosignificant sequence similarity to any previously isolated insecticidaltoxins, and it shows no sequence or structural homology with theomega-atracotoxin-Hv1 family of insecticidal toxins previously isolatedfrom H. versuta (See, Atkinson et al., Insecticidal toxins derived fromfunnel web spider (Atrax or Hadronyche) spiders, PCT/AU93/00039 (WO93/15108) (1993); Fletcher et al., The structure of a novel insecticidalneurotoxin, ω-atracotoxin-HV1, from the venom of an Australian funnelweb spider, Nature Struct. Biol. 4, 559–566 (1997); Wang et al.,Structure-function studies of ω-atracotoxin, a potent antagonist ofinsect voltage-gated calcium channels, Eur. J. Biochem. 264, 488–494(1999)).

The present invention teaches the use of ω-atracotoxin-Hv2a, or the genecoding for the toxin, as a biopesticide, either alone or in combinationwith other insecticidal toxins or genes thereof. It further teaches theuse of the toxin, or the gene coding for the toxin, as a screen fornatural or non-natural compounds that specifically inhibit insectcalcium channels. Furthermore, the present invention provides in thedetermination of the toxin's three-dimensional structure, a model fordeveloping non-peptidic mimics of the toxin that could be used as foliarpesticide sprays.

In a first embodiment of the present invention, there is provided apolypeptide toxin that is toxic to adult and/or larval insects having amolecular mass of approximately 4,478 Daltons and a length of 45 aminoacid residues. The polypeptide is capable of forming three intrachaindisulfide bonds. Activity may be demonstrated by rapid paralysis ofinsects and/or potent inhibition of whole-cell calcium currents inisolated insect neurons. Phylogenetic specificity may typically bedemonstrated by minimal activity in rat or chicken nerve-musclepreparations and/or minimal antagonism of calcium channel currents inisolated rat neurons.

The preferred toxin of the present invention is omega-atracotoxin-Hv2a(SEQ ID NO:1), abbreviated as omega-ACTX-Hv2a or ω-ACTX-Hv2a, as definedherein:

Leu-Leu-Ala-Cys-Leu-Phe-Gly-Asn-Gly-Arg-Cys-Ser-Ser- SEQ ID NO:1Asn-Arg-Asp-Cys-Cys-Glu-Leu-Thr-Pro-Val-Cys-Lys-Arg-Gly-Ser-Cys-Val-Ser-Ser-Gly-Pro-Gly-Leu-Val-Gly-Gly-Ile-Leu-Gly-Gly-Ile-Leu:(LLACLFGNGR CSSNRDCCEL TPVCKRGSCV SSGPGLVGGI LGGIL)

The toxins of the present invention may be isolated from spider venom orchemically synthesized and oxidized/folded using similar techniques tothose described previously for production of syntheticomega-atracotoxin-Hv1a (See, Atkinson et al., Insecticidal toxinsderived from funnel web spider (Atrax or Hadronyche) spiders,PCT/AU93/00039 (WO 93/15108) (1993); Fletcher et al., The structure of anovel insecticidal neurotoxin, ω-atracotoxin-HV1, from the venom of anAustralian funnel web spider, Nature Struct. Biol. 4, 559–566 (1997),both of which are incorporated by reference in their entirety herein).The toxin could also be prepared from a synthetically constructed geneusing recombinant DNA techniques as the authors have describedpreviously for an unrelated protein (Riley et al., Cloning, expression,and spectroscopic studies of the Jun leucine zipper domain, Eur. J.Biochem. 219, 877–886 (1994) which is incorporated in its entiretyherein). A DNA probe coding for the amino sequence of the toxin may beused to isolate the gene coding for the protein or the correspondingpreprotein or preproprotein using standard molecular biologicaltechniques. The natural or synthetic gene(s) may be inserted intoappropriate overexpression vectors for production of the toxin. Inparticular, the gene for the protein, preprotein, or preproprotein maybe inserted into the genome of an appropriate insect vector, such as abaculovirus. Alternatively, transgenic plants may be constructed thatexpress the toxin or the preprotein or preproprotein form of the toxin.Thus, the invention also provides insect viruses and plant speciesengineered to express the toxins of this invention.

In another embodiment of the present invention, there is providedvariants of ω-ACTX-Hv2a, wherein a “variant” is defined as a polypeptidethat corresponds to or comprises a portion of ω-ACTX-Hv2a, or ishomologous to ω-ACTX-Hv2a. For the purposes of this invention,“homology” between two peptide sequences connotes a likeness short ofidentity, indicative of a derivation of the first sequence from thesecond.

In particular, a polypeptide is “homologous” to ω-ACTX-Hv2a if acomparison of their amino acid sequences reveals an identity greaterthan about 30% (which is usually sufficient to indicate structuralhomology). Such a sequence comparison can be performed via numerouscomputer algorithms in the public domain.

In yet another embodiment of the present invention, there is provided amethod of screening for, or designing, an antagonist of insect calciumchannels. This method involves selecting or designing a substance whichinhibits the binding of ω-ACTX-Hv2a, or a variant thereof, to insectcalcium channels and testing the ability of the substance to act as anantagonist of insect calcium channels. The term “insect calcium channel”refers to any insect calcium channel that is inhibited by ω-ACTX-Hv2a.

There is also provided by the present invention, a method of screeningfor substances for insecticidal potency and phylogenetic specificity,the method comprising: (a) measuring the ability of a substance toinhibit the binding of ω-ACTX-Hv2a, or a variant thereof, to insectcalcium channels; (b) measuring the ability of the substance toantagonize insect calcium channels; and (c) determining whether thesubstance has minimal activity against vertebrate calcium channels.Preferably the substance isolated by use of such method has highphylogenetic specificity being defined herein as greater than 100-foldselectivity for insect over vertebrate calcium channels, and preferablygreater than 1000-fold selectivity for insect over vertebrate calciumchannels.

According to yet another embodiment of the present invention there isprovided an insecticidal composition for delivering ω-ACTX-Hv2a, avariant thereof, or an inhibitor of insect calcium channels, discernedby the methods described above. For example, where the toxin, variant,or calcium channel antagonist can be expressed by an insect virus, thevirus encoding the toxin, variant, or calcium channel antagonist can beapplied to the crop to be protected. The virus may be engineered toexpress ω-ACTX-Hv2a, a ω-ACTX-Hv2a variant, or one of the calciumchannel inhibitors either alone, in combination with one another, or incombination with other insecticidal polypeptide toxins that may resultin synergistic insecticidal activity. The virus may be formulated in anagriculturally acceptable carrier, diluent and/or excipient. Suitableviruses include, but are not limited to, baculoviruses.

Alternatively, the crop itself may be engineered to express ω-ACTX-Hv2a,a ω-ACTX-Hv2a variant, or a calcium channel antagonist, discerned by theabove described methods, either alone, in combination, or in combinationwith other insecticidal polypeptide toxins that may result insynergistic insecticidal activity. Crops for which this approach wouldbe useful include cotton, tomato, green bean, sweet corn, lucerne,soybean, sorghum, field pea, linseed, safflower, rapeseed, sunflower,and field lupins.

Alternatively, the insecticidal agent may be delivered directly to thecrop in an agriculturally acceptable carrier, diluent and/or excipient.Delivery could, for example, be in the form of a foliar spray. Insectinfestation of crops may be controlled by treating the crops and/orinsects with such compositions. The insects and/or their larvae may betreated with the composition, for example, by attracting the insects tothe composition with an attractant.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, panel a, is a reverse-phase high performance liquid chromatogramof whole venom isolated from H. versuta with the arrow indicating theretention time of of ω-ACTX-Hv2a.

FIG. 1, panel b, is a reverse-phase high performance liquid chromatogramof ω-ACTX-Hv2a purified from the venom of H. versuta.

FIG. 2 is a reverse-phase high performance liquid chromatogram ofω-ACTX-Hv2a that has been reduced, alkylated with vinylpyridine, thentreated with Staphylococcus aureaus strain V8 type XVII-B protease.

FIG. 3 depicts the primary structure of ω-ACTX-Hv2a as elucidated fromN-terminal and C-terminal amino acid sequencing data.

FIG. 4 is a schematic of the three-dimensional structure of ω-ACTX-Hv2a.

FIG. 5 illustrates the whole-cell calcium current measured in isolatedbee brain neurons exposed to 1 nM and 10 nM ω-ACTX-Hv2a.

FIG. 6 illustrates the time course for the inhibition of whole-cellcalcium channel currents in a bee brain neuron incubated withω-ACTX-Hv2a.

FIG. 7 illustrates dose-response curves for inhibition of whole-cellcalcium currents by ω-ACTX-Hv2a and ω-agatoxin-IVA (from the Americanfunnel-web spider A. aperta) in bee brain and mouse trigeminal neurons.

5. DETAILED DESCRIPTION OF THE INVENTION

There is disclosed an extremely potent and specificpolypeptide-antagonist of insect calcium channels identified by SEQ IDNO:1 and referenced herein as “omega-ACTX-Hv2a” or “ω-ACTX-Hv2a”. Suchantagonist consists of forty-five amino acid residues, has a molecularmass of approximately 4,478 Daltons, and is capable of forming threeintrachain disulfide bonds. Such antagonist has been found to be toxicto both adult and/or larval insects and to display more than 50,000-foldspecificity for invertebrate over vertebrate voltage-gated calciumchannels.

SEQ ID NO:1 may be derived by chemically synthesizing the same andoxidizing/folding the peptide using similar techniques to thosedescribed previously for production of synthetic omega-atracotoxin-Hv1a(See, Atkinson et al., Insecticidal toxins derived from funnel webspider (Atrax or Hadronyche) spiders, PCT/AU93/00039 (WO 93/15108)(1993); Fletcher et al., The structure of a novel insecticidalneurotoxin, ω- atracotoxin-HV1, from the venom of an Australian funnelweb spider, Nature Struct. Biol. 4, 559–566 (1997), both of which areincorporated by reference in their entirety herein). The polypeptideantagonist may also be derived by isolation from spider venom, inparticular the venom of Hadronyche versuta and other Australianfunnel-web spiders of the genera Hadronyche and Atrax.

The polypeptide antagonist SEQ ID NO: 1 may also be derived byconstructing a synthetic gene coding for the polypeptide (e.g., based oncomputer-based back-translation), cloning the gene into an appropriatevector, transforming a cell line with the vector, causing thepolypeptide to be expressed, and purifying the polypeptide. Expressionsystems may contain control sequences, such as promoters, enhancers andtermination controls such as are known in the art for a variety of hosts(See, e.g, Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Ed., Cold Spring Harbor Press (1989) which is incorporated hereinin its entirety). The expression systems may also contain signal peptideand proprotein sequences that facilitate expression of the toxin geneand/or folding of the toxin.

The polypeptide toxins of the present invention may be prepared usingrecombinant DNA techniques such as described in Riley et al., Cloning,expression, and spectroscopic studies of the Jun leucine zipper domain,Eur. J. Biochem 219, 817–886 (1994) (such reference being incorporatedby reference in its entirely herein) which was authored by certain ofthe present inventors.

The polypeptide toxins of the present invention may be prepared in bothprokaryotic and eukaryotic systems. Constructs may be made wherein thecoding sequence for the polypeptide is preceded by an operable signalpeptide which results in secretion of the protein. The particulars forconstruction of expression systems and purification of peptides, andcleavage from fusion peptides are well known to those of ordinary skillin the art. Technology for introduction of DNA into cells includes fourgeneral methods: (1) physical methods such as microinjection,electroporation and the gene gun (See, e.g., Johnston et al., Gene guntransfection of animal cells and genetic immunization, Methods Cell.Biol. 43(A), 353–365 (1994)); (2) viral vectors (See, e.g., Eglitis etal., Retroviral vectors for introduction of genes into mammalian cells,Biotechniques 6(7), 608–614 (1988)); (3) chemical methods (See, e.g.,Ausubel et al., Current Protocols in Molecular Biology, Vol. 1, GreenePublishing Associates/John Wiley & Sons (1993); Zatloukal et al.,Transferrinfection: A highly efficient way to express gene constructs ineukaryotic cells, Ann. N.Y. Acad. Sci. 660, 136–153 (1992)), and (4)receptor-mediated mechanisms (See, e.g., Wagner et al., Coupling ofadenovirus to transferrin-polylysine/DNA complexes greatly enhancesreceptor mediated gene delivery and expression of transfected genes,Proc. Natl. Acad. Sci. USA 89(13), 6099–6103 (1992)). As would beunderstood by one of ordinary skill in the art, minor modification ofthe primary amino acid sequence of SEQ ID NO:1 may result in apolypeptide which has substantially equivalent or enhanced activity ascompared to SEQ ID NO:1. By “modification” of the primary amino acidsequence it is meant to include “deletions” (that is, polypeptides inwhich one or more amino acid residues are absent), “additions” (that is,a polypeptide which has one or more additional amino acid residues ascompared to the specified polypeptide), “substitutions” (that is, apolypeptide which results from the replacement of one or more amino acidresidues), and “fragments” (that is, a polypeptide consisting of aprimary amino acid sequence which is identical to a portion of theprimary sequence of the specified polypeptide). By “modification” it isalso meant to include polypeptides that are altered as a result ofpost-translational events which change, for example, the glycosylation,amidation, lipidation pattern, or the primary, secondary, or tertiarystructure of the polypeptide.

Preferred “substitutions” are those that are conservative, ie., whereinthe residue is replaced by another of the same general type. In makingchanges, the hydropathic index of amino acids may be considered (See,e.g., Kyte. et al., J. Mol. Biol. 157, 105–132 (1982), hereinincorporated by reference in its entirety). It is known in the art thatcertain amino acids may be substituted by other amino acids having asimilar hydropathic index or score and still result in a polypeptidehaving similar biological activity. In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those that are within ±1 are more preferred, and those within±0.5 are even more preferred. Similarly, select amino acids may besubstituted by other amino acids having a similar hydrophilicity, as setforth in U.S. Pat. No. 4,554,101 (herein incorporated by reference inits entirety). In making such changes, as with the hydropathic indices,the substitution of amino acids whose hydrophilicity indices are within±2 is preferred, those that are within ±1 are more preferred, and thosewithin ±0.5 are even more preferred.

Amino acid changes may be achieved by changing the codons of the DNAsequence making use, for example, of known redundancy in the code:

TABLE 1 Three- Single Letter Letter Desig- Desig- Amino Acid nationnation Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGUAspartic Acid Asp D GAC GAU Glutamic Acid Glu E GAA GAG PhenylalaninePhe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAUIsoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUGCUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU ProlinePro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGACGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACCACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr YUAC UAU

Preferably any variant is homologous to ω-ACTX-Hv2a. Sequence comparisoncan be performed via numerous computer algorithms that are well known tothe skilled artisan. An homologous peptide may be produced in accordancewith the present invention, for example, by conventional site-directedmutagenesis (which is one avenue for routinely identifying residues ofthe molecule that are functionally important or not), by random mutation(by “mutation” it is meant an alteration in the primary structure of thepolypeptide due to changes in the nucleotide sequence of the DNA whichencodes it), by chemical synthesis, or by chemical or enzymatic cleavageof ω-ACTX-Hv2a, and other techniques known to those of ordinary skill inthe art.

Recombinant DNA technology can be used to produce a recombinantexpression vector virus of the polypeptide antagonists of the presentinvention. For example, a baculovirus expression vector such as the typedisclosed in U.S. Pat. No. 4,879,236 (which patent is incorporated byreference in its entirety herein) may be produced. Other publicationsdescribing a method for recombinant protein expression using baculovirusvectors include Tomalski, et al., Nature 352, 82–85 (1991), Stewart etal., Nature 352, 85–88 (1991) and McCutchen et al., Biotechnology 9,848–851 (1991). The recombinant expression vector virus could be appliedto the area where the insect is a pest. When the virus is ingested bythe insect its replication will begin. During replication, the gene forthe insecticidally effective protein is expressed, resulting in thedisablement or death of the insect. The virus may express ω-ACTX-Hv2a,or variant thereof, or a calcium channel antagonist discovered bymethods described herein. The virus could also be engineered to expressω-ACTX-Hv2a, a variant thereof, or such calcium channel antagonist inthe various combinations possible with one another, and furthermore incombination with other insecticidal polypeptide toxins. Suchcombinations may result in synergistic insecticidal activity. Hybridbacterial cells, comprising a plasmid with the gene coding forpolypeptide antagonists of the present invention may likewise be used tocontrol insects in conformity with the present invention.

Insect calcium channel antagonists, viral vectors, and hybrid bacterialcells of the present invention may be applied in the form of a foliarspray comprising an agriculturally acceptable carrier. Crops for whichthis approach would be useful are numerous, and include, withoutlimitation, cotton, tomato, green bean, sweet corn, lucerne, soybean,sorghum, field pea, linseed, safflower, rapeseed, sunflower, and fieldlupins. Such agents may also be applied to insects directly.

As would be understood by one of ordinary skill in the art, plants maybe produced that express the polypeptide antagonists of the presentinvention. By “transgenic plant” it is meant any plant, or progenythereof, derived from a “transformed plant” cell or protoplast, whereinthe plant DNA (nuclear or chloroplast) contains an introduced exogenousDNA molecule not originally present in a native, non-transgenic plant ofthe same strain. Typical vectors useful for expression of genes inhigher plants are well known in the art and include vectors derived fromthe tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens (Rogers etal., Methods in Enzymol. 153, 253–277 (1987)), and pCaMVCN transfercontrol vector (available from Pharmacia, Piscataway, N.J.). Of course,as would be understood by one of ordinary skill in the art, other meansof gene introduction into the cell may also be employed, such aselectroporation (Fromm et al., Expression of genes transferred intomonocot and dicot plant cells by electroporation, Proc. Natl. Acad. Sci.USA 82(17),5824–5828 (1985)), polyethyleneglycol-mediated transformationof protoplasts (Ominrulleh et al., Plant Molecular Biology 21, 415–428(1993)), desiccation/inhibition-mediated DNA uptake, agitation withsilicon carbide fibers, by acceleration of DNA coated particles,injection into reproductive organs, and injection into immature embryos.

If an expression vector of the present invention is used to transform aplant, it is preferred that a promoter be selected that has the abilityto drive expression in the plant. Promoters that function in plants arewell known in the art. Exemplary tissue-specific promoters are cornsucrose synthetase 1 (Yang et al., Proc. Natl. Acad. Sci. USA 87,4144–4148 (1990)), cauliflower mosaic virus (CaMV 35S) promoter, S-E9small subunit RuBP carboxylase promoter, and corn heat shock protein(Odell et al., Nature 335, 810 (1985)). The choice of which expressionvector, and ultimately to which promoter a polypeptide coding region isoperatively linked, depends directly on the functional propertiesdesired, for example, the location and timing of protein expression andthe host cell to be transformed. In a preferred embodiment, the vectorused to express the polypeptide includes a selection marker that iseffective in a plant cell. Transformation vectors used to transformplants and methods of making those vectors are described, for example,in U.S. Pat. Nos. 4,971,908, 4,940,835, 4,769,061 and 4,757,011, thedisclosures of which are incorporated in their entirety herein byreference.

The present invention also encompasses DNA sequences encoding for SEQ IDNO:1 and variants thereof. The DNA sequences encoding for such activepolypeptide sequences allow for the preparation of relatively short DNA(or RNA) sequences having the ability to specifically hybridize to suchgene sequences. The short nucleic acid sequences may be used as probesfor detecting the presence of complementary sequences in a given sample,or may be used as primers to detect, amplify or mutate a defined segmentof the DNA sequences encoding for SEQ ID NO:1, and variants thereof. Apreferred nucleic acid sequence employed for hybridization studies is atleast 14 nucleotides in length to ensure that the fragment is ofsufficient length to form a stable and selective duplex molecule. Suchfragments may be prepared by, for example, directly synthesizing thefragment by chemical means, by application of nucleic acid reproductiontechnology, such as the PCR technology (described in U.S. Pat. Nos.4,683,195 and 4,683,202, herein incorporated in their entirety byreference), or by excising selected nucleic acid fragments formrecombinant plasmids containing appropriate inserts and suitablerestriction sites.

Improved methods for screening for and/or designing antagonists ofinsect calcium channels are also provided. Given the large differencebetween the binding constants of ω-ACTX-Hv2a with respect toinvertebrate versus vertebrate calcium channels, in particular thosecalcium channels associated with the insect neuronal system, ω-ACTX-Hv2amay be effectively used in screening procedures to identify newantagonists of insect calcium channels. Using conventionalstructure-activity analysis of identified insect calcium channelantagonists, routes of chemical design of other more potent antagonistsmay be pursued based on the identified antagonists.

One method for isolating compounds having insect calcium channelantagonist activity comprises the steps of: preparing an invertebratecellular preparation having a substantial number of unbound calciumchannels; adding an amount of ω-ACTX-Hv2a effective to substantiallybind all of the calcium channels of the invertebrate cellularpreparation; selecting a test compound; adding the test compound to theinvertebrate cellular preparation bound with ω-ACTX-Hv2a; measuring theamount of ω-ACTX-Hv2a released by the addition of the test compound.

Another method for isolating compounds having insect calcium channelantagonist activity comprises the steps of: preparing an invertebratecellular preparation having a substantial number of unbound calciumchannels; selecting a test compound; adding the test compound to theinvertebrate cellular preparation at a set concentration; allowing thetest compound to incubate for a period of time sufficient to allow thetest compound to bind with the calcium channels of the preparation;washing the invertebrate cellular preparation which was incubated withthe test compound so as to remove any unbound test compound; adding anamount of ω-ACTX-Hv2a sufficient to bind all of the calcium channels ofthe untreated invertebrate cellular preparation; measuring the amount oftest compound displaced by the addition of the ω-ACTX-Hv2a.

Preferably, any antagonist identified by the screening procedure willbind strongly to insect calcium channel(s) such that the dissociationconstant (K_(d)) for its interaction with the calcium channel(s) is lessthan 10⁻⁷ M and more preferably less than 10⁻⁹ M. Preferably, theactivity of the test compound against vertebrate calcium channels isthen determined so as to discern the relative selectivity of thecompound. In a preferred embodiment, phylogenetic specificity is greaterthan 100-fold for insect over vertebrate calcium channels, and morepreferably greater than 1000-fold. As would be recognized by one ofordinary skill in the art, other types of competitive assays andpharmacological activity screening procedures known in the art may beadapted to utilize ω-ACTX-Hv2a to provide for improved screening ofcompounds for invertebrate calcium channel antagonism.

Test compounds may comprise a compound from an archive of naturalcompounds or from combinatorial libraries of peptidic and non-peptidiccompounds.

Libraries of mutated toxins for the purposes of screening may beobtained by in vitro evolution of a gene for ω-ACTX-Hv2a as describedpreviously for unrelated proteins (Stemmer, DNA shuffling by randomfragmentation and re-assembly; in vitro recombination for molecularevolution, Proc. Natl. Acad. Sci. 91, 10747–10751 (1994); Stemmer, Rapidevolution of a protein in vitro by DNA shuffling, Nature 370, 389–391(1994); Zhang et al., Directed evolution of a fucosidase from aglactosidase by DNA shuffling and screening, Proc. Natl. Acad. Sci. USA94, 4504–4509 (1997), all of which are incorporated by reference intheir entirety herein). This could be done using error-prone PCR of theentire ω-ACTX-Hv2a gene or digestion of the ω-ACTX-Hv2a gene with anappropriate enzyme followed by error-prone PCR reconstruction of theentire gene sequence. These error-prone PCR procedures could also beapplied to the complete preproprotein gene sequence for ω-ACTX-Hv2a. Thelibrary of mutant ω-ACTX-Hv2a gene sequences could then be used togenerate a series of ω-ACTX-Hv2a variant antagonists. These antagonistsmay then be screened for their ability to inhibit the binding ofω-ACTX-Hv2a, or a variant thereof, to insect calcium channels, ordirectly for their ability to inhibit insect calcium channels. Screeningmay be performed, for example, by phage display of a mutant gene libraryfollowed by selection of phage particles that bind tightly to insectcalcium channels, or phage particles that inhibit the binding ofω-ACTX-Hv2a, or a variant thereof, to insect calcium channels. As wouldbe understood by one of ordinary skill in the art, a mutant gene librarycould also be constructed by other standard molecular biological methodssuch as oligonucleotide cassette mutagenesis or construction ofsynthetic genes with certain nucleotide positions randomised.

The three-dimensional structure of ω-ACTX-Hv2a, which has beenelucidated by the present inventors as set forth below, may also be usedto search structure libraries for (or to design) either peptidic ornon-peptidic compounds that resemble the key structural elements ofω-ACTX-Hv2a, particularly those regions found to be critical foractivity by mutagenesis/truncation/modification experiments. Thesecompounds could then be tested for their ability to inhibit the bindingof ω-ACTX-Hv2a, or a variant thereof, to insect calcium channels.

The ω-ACTX-Hv2a (or variant thereof) used in a competitive assay may beradioactive or fluorescently labeled, all of which fall within the scopeof the present invention. Screening of test compounds may be performedusing either native or recombinantly produced calcium channels, orstructurally-modified calcium channels.

The present inventors have found that acutely isolated cockroach and beebrain neurons are particularly suitable to provide the basis for asensitive electrophysiological assay for assaying substances thatinterfere with the ability of ω-ACTX-Hv2a to inhibit insect calciumchannels, while a variety of mouse sensory ganglion neurons are suitableto provide the basis for a sensitive electrophysiological assay fortesting the ability of compounds to inhibit vertebrate calcium channels.It will be appreciated, however, that other insect and vertebrate cellsor cell lines would also be suitable for use in this aspect of thepresent invention. For example, transient expression of cloned insectcalcium channels in suitable cell lines or oocytes could form the basisof a suitable assay system.

Now turning to several examples that illustrate particular compositionsand methods within the scope of the present invention. Such examples,and the figures associated therewith, are presented in order to makecertain aspects of the present invention more clearly understood, andare not intended to limit the scope of the invention as described hereinin any manner.

EXAMPLE 1 Elution and Purification of Polyleptide Toxin from H. versutaWhole Venom

Lyophilized crude venom (1.25 mg) was dissolved in 50 μl distilledwater, loaded onto a Vydac C18 analytical reverse-phase HPLC column(4.6×250 mm, 5 μm pore size), and eluted at a flow rate of 1 ml min⁻using a gradient of 5–25% Buffer B (0.1% TFA in acetonitrile) over 22min, followed by a gradient of 25–50% Buffer B over 48 min. Buffer A was0.1% TFA in water. FIG. 1 depicts two reverse-phase HPLC chromatograms.FIG. 1 a is a reverse-phase HPLC chromatogram of the whole venomisolated from H. versuta. The elution position of the polypeptide toxinreferred to as ω-ACTX-Hv2a (retention time of approximately 48 min) ismarked with an arrow. FIG. 1 b is a reverse-phase HPLC chromatogram ofthe ω-ACTX-Hv2a that had been purified from H. versuta venom usingstandard chromatographic purification techniques.

EXAMPLE 2 Determination of Primary Structure of ω-ACTX-Hv2a

Isolated ω-ACTX-Hv2a (50 μg) was reduced and pyridylethylated, thendigested with Staphylococcus aureus strain V8 type XVII-B protease [EC3.4.21.19] for 6 hours at a toxin:protease ratio of 100:1 and atemperature of 37° C. The reaction was carried out in 50 mM Tris buffer,pH 7.8. The resulting peptide fragments (labeled V1–V4) were applied toa Vydac C18 analytical reverse-phase HPLC column (4.6×250 mm, 5 μm poresize), and eluted at a flow rate of 1 ml min⁻¹ using a gradient of 5–60%Buffer B (0.1% TFA in acetonitrile) over 40 min. Buffer A was 0.1% TFAin water. FIG. 2 shows a reverse-phase HPLC chromatogram of thefragments resulting from digestion of ω-ACTX-Hv2a.

The primary structure of ω-ACTX-Hv2a was reconstructed from N-terminaland C-terminal sequencing of the complete toxin as well as sequencing ofvarious fragments obtained from digestion with V8 protease. The primarystructure is shown in FIG. 3 using the internationally recognizedone-letter abbreviations for each of the amino acids. Such structure asshown was reconstructed from N-terminal and C-terminal sequencing aswell as V8 fragment 4 obtained by digestion with V8 protease. Thedisulfide-bonding pattern of ω-ACTX-Hv2a, as determined from thethree-dimensional structure (see FIG. 4), is indicated by the heavylines.

EXAMPLE 3 Determination of Three-Dimensional Structure of ω-ACTX-Hv2a

The three-dimensional structure of ω-ACTX-Hv2a was determined usingstandard two-dimensional homonuclear nuclear magnetic resonance (NMR)spectroscopy techniques familiar to those skilled in the art (see,Fletcher et al., The structure of a novel insecticidal neurotoxin,ω-ACTX-Hv1, from the venom of an Australian funnel web spider, NatureStruct. Biol. 4, 559–566, 1997; Wüthrich, NMR of Proteins and NucleicAcids (John Wiley & Sons, Inc., New York, 1986), both of which areincorporated in their entirety herein). FIG. 4 is a schematic of thethree-dimensional solution structure of residues 1–32 of ω-ACTX-Hv2a;residues 33–45 have no preferred conformation in solution. The structurecontains a 3₁₀ helix encompassing residues 13–17, an antiparallelβ-hairpin comprising two β-strands (β strand 1=residues 23–25, β strand2=residues28–30), and three disulphide bonds (Cys4-Cys18, Cys11-Cys24,and Cys17-Cys29). These structural features are all delineated by arrowsin FIG. 4.

EXAMPLE 4 Effect of ω-ACTX-Hv2a on Whole-Cell Calcium Channel CurrentsIn Isolated Bee Neurons

Neurons were isolated from the brains of adult European honey bees, Apismelliferaa, as the authors have described previously (see, Wang et al.,Discovery and characterization of a family of insecticidal neurotoxinswith a rare vicinal disulfide bond, Nature Struct. Biol. 7, 505–513,which is incorporated in its entirety herein). Standard whole cellvoltage clamp recordings were made of bee brain calcium channel(I_(Ca)), sodium channel (I_(Na)) and potassium channel (I_(K)) currentsat ambient temperature (22–24° C.). For bee neurons, recordings weremade with fire polished borosilicate pipets of ˜6 M resistance whenfilled with intracellular solution of either of the followingcompositions (mM): CsCl 120, NaCl 5, MgATP 5, Na₂GTP 0.3, EGTA 10, CaCl₂2 and HEPES 10, pH 7.3 (for I_(Na) and I_(Ca)) or KF 130, EGTA 10, CaCl₂2 and HEPES 10, pH 7.3 for recording I_(K). For recordings of I_(Ca) andI_(Na) the external solution consisted of NaCl 135, tetraethylammoniumchloride (TEACl) 20, CsCl 5, BaCl₂ 5, HEPES 10, glucose 10, BSA 0.05%,pH 7.3. For I_(K) recording the external solution consisted of (mM) NaCl130, KCl 20, CaCl₂ 2.5, MgCl₂ 1.5, HEPES 10, glucose 10, BSA 0.05%, pH7.3.

Neurons were voltage clamped at −90 mV and currents evoked by steppingthe membrane potential from −60 to +60 mV. Toxin effects on I_(Ca) andI_(Na) were tested at the potential with largest inward current, usually−10 or 0 mV. In bee neurons the peak inward currents were usuallyabolished by 100 μM Cd²⁺, suggesting that the current was largelycarried by Ca²⁺ channels. In a few bee neurons there was a rapidlyactivating, transient and Cd²⁺-insensitive current which was blockedcompletely by tetrodotoxin (TTX, 1 μM).

FIG. 5 illustrates the effect of ω-ACTX-Hv2a on whole-cell calciumchannel currents in such isolated bee brain neurons. The figure showsthe whole-cell calcium current obtained from a bee brain neuron in theabsence (“control”) or presence of 1 nM or 10 nM ω-ACTX-Hv2a.Application of ω-ACTX Hv2a (10 pM to 100 nM) inhibited calcium channelcurrents in all neurons examined (n=37). The almost complete abrogationof calcium channel currents by these low concentrations of toxinindicates that most, if not all, bee brain calcium channels aresensitive to ω-ACTX-Hv2a. This contrasts with ω-ACTX-Hv1a, whichinhibits whole-cell calcium channel currents in isolated cockroachneurons by only 25±10% at a concentration of 100 nM (see FIG. 6 inFletcher et al., The structure of a novel insecticidal neurotoxin,ω-ACTX-Hv1, from the venom of an Australian funnel web spider, NatureStruct. Biol. 4, 559–566, 1997).

FIG. 6 illustrates the time course for the inhibition of whole-cellcalcium channel currents in a bee brain neuron following addition of 1nM and 10 nM ω-ACTX-Hv2a: the effect is rapid and only very slowlyreversible as indicated by the protracted recovery of channel activityafter initiating a wash step (indicating by the solid horizontal line).The rapid calcium channel inhibition and slow recovery observed in theseelectrophysiological experiments is consistent with the phenotypiceffects observed when the toxin is injected into house crickets (Achetadomesticus Linnaeus). Injection into crickets causes immediate paralysiswith a PD₅₀ (the dose required to paralyse 50% of injected insects) of160±9 pmol g⁻¹ and a mean paralysis time of 4–5 h at a dose of 250–500pmol g⁻¹. Injection of crickets with a second dose (250–500 pmol g⁻¹) oftoxin prior to reversal of paralysis was lethal. In striking contrast,the toxin did not provoke any adverse effects when injectedsubcutaneously into newborn BALB/c mice (3.1±0.2 g, n=3) at doses up to800 pmol g⁻¹, which is 5-fold higher than the PD₅₀ in crickets.

EXAMPLE 5 Comparison of the Effects of ω-ACTX-Hv2a and ω-agatoxin-IVA onWhole-Cell Calcium Channel Currents In Isolated Bee Neurons and MouseTrigeminal Neurons

Bee brain neurons were isolated as described in Example 4 above. Mousetrigeminal ganglion neurons were isolated by gentle trituration ofminced ganglia following a 20-min treatment at 34 C with papain (20units ml⁻¹) in a HEPES buffered saline (HBS) solution of composition (inmM): NaCl 140, KCl 2.5, CaCl₂ 2.5, MgCl₂ 1.5, HEPES 10, glucose 10, pH7.3. Standard whole cell voltage clamp recordings were made of bee braincalcium channel (I_(Ca)), sodium channel (I_(Na)) and potassium channel(I_(K)) currents and mouse sensory neuron I_(Ca) and I_(Na) at ambienttemperature (22–24 C). The same internal solution as described for thebee brain recordings in Example 4 was used for recordings of mousesensory neuron I_(Ca) and I_(Na); the electrodes had a resistance of 1–2M. The I_(Ca) external solution for the mouse neuron recordingscontained (mM): TEACl 140, CaCl2 2.5, CsCl 2.5, HEPES 10, glucose 10,BSA 0.05%, pH 7.3, while I_(Na), were recorded in HBS.

Neurons were voltage clamped at −90 mV and currents evoked by steppingthe membrane potential from −60 to +60 mV. In mouse sensory neurons, thepeak inward currents evoked in the presence of potassium and sodiumchannel blockers were abolished by 30 μM Cd²⁺. The inward currentsrecorded in HBS consisted of both TTX-sensitive and TTX-resistantcomponents. Toxin effects on bee brain I_(K) were determined over arange of membrane potentials (from −40 to +60 mV). Data were collectedand analysed as described previously (see, Fletcher et al., Thestructure of a novel insecticidal neurotoxin, ω-ACTX-Hv1, from the venomof an Australian funnel web spider, Nature Struct. Biol. 4, 559–566,1997).

FIG. 7 shows that the EC₅₀ for ω-ACTX-Hv2a inhibition of I_(Ca) was ˜150pM (see data indicated by filled circles), with maximum inhibitionoccurring at concentrations >10 nM. Application of the Americanfunnel-web spider toxin ω-agatoxin-IVA (ω-Aga-IVA; see filled squares inFIG. 7), the prototypic antagonist of vertebrate P-type voltage-gatedcalcium channels (see, Mintz et al., P-type calcium channels blocked bythe spider toxin omega-Aga-IVA, Nature 355, 827–829, 1992), alsoinhibited I_(Ca) in all bee neurons examined (n=19), but the EC₅₀ (10nM) and the concentration required for maximum inhibition (>100 nM) wereboth significantly higher than for ω-ACTX-Hv2a.

In striking contrast, superfusion of high concentrations of ω-ACTX-Hv2a(1 μM, n 10) for 5 min had virtually no effect on I_(Ca) in mousesensory neurons (see unfilled circles in FIG. 7), whereas application ofω-Aga-IVA inhibited a component of I_(Ca) in all mouse sensory neuronswith an EC₅₀ of about 20 nM (maximum I_(Ca) inhibition ˜40%; seeunfilled squares in FIG. 7). ω-ACTX-Hv2a (100 nM) did not inhibit theTTX-sensitive I_(Na) of bee brain neurons (I_(Na) was 98±4% of control,n=4), nor did it significantly affect I_(Na) in mouse sensory neurons(I_(Na) was 97±3% of control with ω-ACTX-Hv2a =1 μM, n=5). ω-ACTX-Hv2a(10 nM, n=1; 100 nM, n=5) had no effect on bee brain I_(K) at anypotential when neurons were stepped from −90 mV to between −40 and +60mV.

It may be concluded that ω-ACTX-Hv2a is a potent and extremely specificantagonist of insect voltage-gated calcium channels. The toxin has noeffect on potassium and sodium currents in either bee brain or mousetrigeminal neurons. Based on the data presented in FIG. 7, ω-ACTX-Hv2amay be calculated as having >50,000-fold preference for insect versusvertebrate calcium channels, making it >25,000-fold more selective thanω-agatoxin-IVA (which only has a 2-fold preference). Thus, ω-ACTX-Hv2ais one of the most invertebrate-selective peptide toxins discovered todate.

While the invention has been described with respect to certainembodiments, those skilled in the art will readily appreciate thatvarious changes and/or modifications can be made to the inventionwithout departing from the scope of the invention, and such changesand/or modifications are to be included within the spirit and purview ofthis application and the scope of the appended claims.

1. A polypeptide which binds calcium channels of invertebrate pestswhich is substantially non-toxic against vertebrates, comprising apolypeptide isolated from Hydronyche versuta having a molecular mass ofapproximately 4,400 to 4,500 Daltons, and comprising about 45 aminoacids.
 2. The polypeptide of claim 1, wherein the polypeptide furthercomprises the capacity to form three intrachain disulfide bonds.